Tài liệu Báo cáo khoa học: ER stress and diseases docx

REVIEW ARTICLE
ER stress and diseases
Hiderou Yoshida
1,2
1 Department of Biophysics, Graduate School of Science, Kyoto University, Japan
2 PRESTO-SORST, Japan Science and Technology Agency, Japan
Keywords
conformational disease; cytoplasmic splicing;
ER stress response; ER-associated protein
degradation (ERAD); Golgi stress response
Correspondence
H. Yoshida, Department of Biophysics,
Graduate School of Science, Kyoto
University, Kitashirakawa-Oiwakecho,
Sakyo-ku, Kyoto 606-8502, Japan
Fax: +81 75 753 3718
Tel: +81 75 753 4201
E-mail:
kyoto-u.ac.jp
(Received 11 September 2006, revised
14 November 2006, accepted 8 December
2006)
doi:10.1111/j.1742-4658.2007.05639.x
Proteins synthesized in the endoplasmic reticulum (ER) are properly folded
with the assistance of ER chaperones. Malfolded proteins are disposed of
by ER-associated protein degradation (ERAD). When the amount of
unfolded protein exceeds the folding capacity of the ER, human cells acti-
vate a defense mechanism called the ER stress response, which induces
expression of ER chaperones and ERAD components and transiently
attenuates protein synthesis to decrease the burden on the ER. It has been
revealed that three independent response pathways separately regulate

induction of the expression of chaperones, ERAD components, and trans-
lational attenuation. A malfunction of the ER stress response caused by
aging, genetic mutations, or environmental factors can result in various dis-
eases such as diabetes, inflammation, and neurodegenerative disorders
including Alzheimer’s disease, Parkinson’s disease, and bipolar disorder,
which are collectively known as ‘conformational diseases’. In this review, I
will summarize recent progress in this field. Molecules that regulate the ER
stress response would be potential candidates for drug targets in various
conformational diseases.
Abbreviations
AIGP, axotomy-induced glyco ⁄ Golgi protein; APP, amyloid precursor protein; ASK1, apoptosis signal-regulating kinase 1; ATF, activating
transcription factor; BAK, Bcl-2 homologous antagonist ⁄ killer; BAP, BiP-associated protein; Bap31, B cell receptor-associated protein 31; Bax,
Bcl2-associated X protein; Bcl2, B cell leukemia 2; BI-1, Bax inhibitor 1; Bim, Bcl2-interacting mediator of cell death; BiP, binding protein; bZIP,
basic leucine zipper; c-Abl, Abelson murine leukemia viral oncogene homolog 1; C ⁄ EBP, CCAAT ⁄ enhancer-binding protein; CHOP, C ⁄ EBP-
homologous protein; CREB, cAMP response element-binding protein; CREBH, cAMP response element-binding protein H; CReP, constitutive
repressor of eIF2a phosphorylation; DAP, death-associated protein; Der1, degradation in the endoplasmic reticulum protein 1; Derlin-1, Der1-
like protein 1; Doa10, degradation in the endoplasmic reticulum protein 10; DR5, death receptor 5; EDEM, ER degradation enhancing
a)mannosidase-like protein; eIF2 a, a-subunit of eukaryotic translational initiation factor 2; ER, endoplasmic reticulum; ERAD, ER-associated
degradation; ERdj, ER dnaJ; ERO1, ER oxidoreductin; ERp72, ER protein 72; ERSE, ER stress response element; FKBP13, FK506-binding
protein 13; GADD, growth arrest and DNA damage; gp78, glycoprotein 78; GRP, glucose-regulated protein; HEDJ, human ER-associated dnaJ;
HIAP2, human inhibitor of apoptosis 2; HRD1, HMG-CoA reductase degradation protein 1; HSP, heat shock protein; IAP, inhibitor of apoptosis;
IDDM, insulin-dependent diabetes mellitus; IRE1, inositol requirement 1; JNK, Jun kinase; Keap1, Kelch-like Ech-associated protein 1; LZIP,
basic leucine zipper protein; NIDDM, noninsulin-dependent diabetes mellitus; NOXA, neutrophil NADPH oxidase factor; Npl4, nuclear protein
localization 4; NRF, nuclear respiratory factor; ORP150, oxygen-regulated protein 150; OS9, osteosarcoma 9; p58IPK, 58 kDa-inhibitor of
protein kinase; pATF6(N), the nuclear form of ATF6 protein; PDI, protein disulfide isomerase; PERK, PRKR-like endoplasmic reticulum kinase;
PKR, double stranded RNA-dependent protein kinase; PLP1, proteolipid protein 1; polyQ, polyglutamine; PrP, pion protein; PrP
c
, cellular PrP;
PrP
Sc

, scrapie PrP; PS1, presenillin 1; PUMA, p53 up-regulated modulator of apoptosis; pXBP1(S), the spliced form of XBP1 protein; pXBP1(U),
the unspliced form of XBP1 protein; RIP, regulated intramembrane proteolysis; RseA, regulator of s
E
; S1P, site 1 protease; S2P, site 2
protease; SAPK, stress-activated protein kinase; SEL1, suppressor of lin12-like; SREBP, sterol response element-binding protein; TDAG51,
T cell death-associated gene 51; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TRAF2, TNF receptor-associated
factor 2; TRB3, Tribbles homolog 3; UBC6, ubiquitin conjugase 6; UBC7, ubiquitin conjugase 7; UBE1, ubiquitin-activating enzyme 1; UBE2G2,
ubiquitin-activating enzyme 2G2; UBX2, UBX domain-containing protein 2; UCH-L1, ubiquitin C-terminal esterase L1; Ufd1, ubiquitin fusion
degradation protein 1; UPRE, unfolded protein response element; VCP, valocin-containing protein; WFS1, Wolfram syndrome 1; XBP1, x-box
binding protein 1; XIAP, inhibitor of apoptosis, x-linked; XTP3B, XTP3-transactivated gene B.
630 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS
Introduction
The endoplasmic reticulum (ER) is an organelle where
secretory or membrane proteins are synthesized. Nas-
cent proteins are folded with the assistance of molecu-
lar chaperones and folding enzymes located in the ER
(collectively called ER chaperones), and only correctly
folded proteins are transported to the Golgi apparatus
(Fig. 1). Unfolded or malfolded proteins are retained
in the ER, retrotranslocated to the cytoplasm by the
machinery of ER-associated degradation (ERAD), and
degraded by the proteasome. ER chaperones and
ERAD components are constitutively expressed in the
ER to deal with nascent proteins. When cells synthes-
ize secretory proteins in amounts that exceed the capa-
city of the folding apparatus and ERAD machinery,
unfolded proteins are accumulated in the ER. Unfol-
ded proteins expose hydrophobic amino-acid residues
that should be located inside the protein and tend to
form protein aggregates. Protein aggregates are so

toxic that they induce apoptotic cell death and cause
‘conformational diseases’ such as neurodegenerative
disorders and diabetes mellitus. To alleviate such a
stressful situation (ER stress), eukaryotic cells activate
a series of self-defense mechanisms referred to collec-
tively as the ER stress response or unfolded pro-
tein response [1–4].
The mammalian ER stress response consists of four
mechanisms. The first is attenuation of protein synthe-
sis, which prevents any further accumulation of un-
folded proteins. The second is the transcriptional
induction of ER chaperone genes to increase folding
capacity, and the third is the transcriptional induction
of ERAD component genes to increase ERAD ability.
The fourth is the induction of apoptosis to safely dis-
pose of cells injured by ER stress to ensure the survival
of the organism.
In this article, I will describe the basics of the mam-
malian ER stress response that are essential to under-
standing conformational diseases. I will review hot
topics such as ERAD, regulated intramembrane pro-
teolysis (RIP) and cytoplasmic splicing, and briefly
summarize the ER stress-related diseases.
ER stress-inducing chemicals
Chemicals such as tunicamycin, thapsigargin, and
dithiothreitol are usually used to evoke ER stress in
cultured cells or animals for experimental purposes. I
will briefly summarize the ER stress-inducing chemicals
below.
The first group of ER stressors comprises glycosyla-

tion inhibitors. Most of the proteins synthesized in
the ER are N-glycosylated, and the N-glycosylation is
cytoplasm
ER
ER chaperone
degraded
ribosome
mRNA
unfolded protein
aggregation
ER stresss
nascent protein
Golgi apparatus
apoptosis folding disease
ERAD
translational attenuation
Fig. 1. Mammalian ER stress response. An accumulation of unfolded proteins in the ER evokes ER stress, and cells induce the ER stress
response to cope. The mammalian ER stress response consists of four mechanisms: (1) translational attenuation; (2) expression of ER chap-
erones; (3) enhanced ERAD; (4) apoptosis.
H. Yoshida ER stress and diseases
FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 631
often essential for protein folding. Thus, chemicals that
disturb N-glycosylation have the potential to induce
ER stress. Tunicamycin is an antibiotic produced by
Streptomyces lysosuperificus that inhibits N-glycosyla-
tion by preventing UDP-GlcNAc–dolichol phosphate
GlcNAc-phosphate transferase activity [5,6]. 2-Deoxy-
d-glucose is also used to inhibit N-glycosylation [7],
but is less efficient than tunicamycin.
Another class of ER stressors is Ca

2+
metabolism
disruptors. As the concentration of Ca
2+
ion in the
ER is kept at a high level and ER chaperones such as
BiP require Ca
2+
ions, chemicals that perturb Ca
2+
metabolism in the ER induce ER stress. Ca
2+
ionoph-
ores such as A23187 and the Ca
2+
pump inhibitor,
thapsigargin, are often used to evoke ER stress [5,8].
The third category of ER stressors is reducing
agents. As the lumen of the ER is highly oxidative,
proteins synthesized there can form intermolecular or
intramolecular disulfide bonds between their cysteine
residues. As the formation of disulfide bonds is
important for the folding of secretory proteins, redu-
cing agents that disrupt disulfide bonds evoke ER
stress. Dithiothreitol and 2-mercaptoethanol are often
used to this end [9,10].
Hypoxia is also known to induce ER stress,
although the underlying mechanism is unknown. It is
speculated that a decrease in glucose concentration
induced by hypoxia (because hypoxia induces glyco-

lytic enzymes to sustain ATP production and then cells
consume glucose) inhibits N-glycosylation, leading to
ER stress [11].
ER chaperones
ER chaperones include molecular chaperones and fold-
ing enzymes located in the ER, which are responsible
for the folding of nascent proteins [4,12]. They are also
involved in the unfolding of malfolded proteins in
ERAD. In this section, I will review mammalian ER
chaperones, focusing on recent discoveries.
Binding protein (BiP) ⁄glucose-regulated protein (GRP)78
is a well-known ER chaperone that belongs to the heat
shock protein (HSP)70 family. BiP binds to the hydro-
phobic region of unfolded proteins via a substrate-
binding domain and facilitates folding through
conformational change evoked by the hydrolysis of
ATP by the ATPase domain. Oxygen-regulated pro-
tein (ORP)150 ⁄ GRP170 is an ER chaperone belonging
to the HSP110 family (a HSP70 subfamily), and facili-
tates protein folding via a mechanism similar to
that for BiP. It was originally identified as a pro-
tein expressed in response to hypoxia. ER dnaJ
(ERdj)1, ERdj3 ⁄ human ER-asociated dnaJ (HEDJ),
ERdj4, ERdj5, SEC63, and p58IPK are ER chaper-
ones belonging to the HSP40 family, and modulate the
functions of BiP by regulating its ATPase activity as a
cochaperone. BiP-associated protein (BAP), which is a
member of the GrpE family, also modulates the func-
tions of BiP by enhancing nucleotide exchange.
GRP94 is an ER chaperone belonging to the HSP90

family, and facilitates folding through the hydrolysis
of ATP. FKBP13 is a peptidyl-prolyl isomerase
belonging to the FKBP family. These ER chaperones
are involved in the general folding process of secretory
proteins.
Calnexin and calreticulin are ER chaperones specif-
ically involved in the folding of glycoprotein. High-
mannose type oligosaccharide is attached en bloc to
most proteins synthesized in the ER, and then trimmed
sequentially (Fig. 2). When two glucose residues are
trimmed by glucosidase I or II and the protein con-
tains only one glucose residue, calnexin and calreticulin
bind and fold the client protein. When the last glucose
residue is trimmed by glucosidase II, the client is
released from calnexin and calreticulin, and binds to
UDP-glucose–glycoprotein glucosyltransferase. If the
protein is folded, it is released from the enzyme and
transported to the Golgi apparatus. If it is not folded,
UDP-glucose–glycoprotein glucosyltransferase attaches
one glucose residue and returns it to calnexin and cal-
reticulin. This folding process is called the calnexin
cycle [13]. Calnexin and calreticulin share a similar
molecular structure and function, although they are
transmembrane and luminal proteins, respectively.
Numerous folding enzymes are involved in the forma-
tion of disulfide bonds in the ER, such as protein disul-
fide isomerase (PDI), ERp72, ERp61, GRP58 ⁄ ERp57,
ERp44, ERp29, and PDI-P5. These folding enzymes
oxidize cysteine residues of nascent proteins and help
proteins to form correct disulfide bonds. Reduced fold-

ing enzymes are reoxidized by ER oxidoreductin
(ERO1), which can use molecular oxygen as a terminal
electron acceptor [14].
ERAD
Unfolded or malfolded proteins are trapped by the
ERAD machinery and transported to the cytoplasm
[15–17]. Retrotranslocated proteins are ubiquitinated
and degraded by the proteasome in the cytosol.
Thus, the process of ERAD can be divided into four
steps, recognition, retrotranslocation, ubiquitination,
and degradation (Fig. 3). As ERAD is one of the
hottest topics in the study of ER stress, I will sum-
marize our current understanding of mammalian
ERAD systems.
ER stress and diseases H. Yoshida
632 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS
Recognition
During the calnexin cycle, the oligosaccharide of nascent
polypeptides contains nine mannose residues. When one
mannose residue is trimmed by a-mannosidase I, nas-
cent polypeptides with eight mannose residues are
released from calnexin or calreticulin and bind to
ER degradation-enhancing a-mannosidase-like pro-
tein (EDEM) (Fig. 2), which discriminates unfolded
proteins from folded proteins [18–22]. There are three
genes for EDEM, and both EDEM1 and EDEM2
are involved in ERAD. EDEM1 is an ER membrane
protein, whereas EDEM2 and EDEM3 are luminal pro-
teins [23–25]. All EDEMs contain the mannosidase-like
Fig. 3. Mammalian ERAD machinery. Unfolded proteins released from the calnexin cycle are captured by a recognition complex containing

EDEM and OS9, moved to the cytosol through retrotranslocation machinery, polyubiquitinated by the E1–E2–E3 system, and degraded by
the proteasome. The precise function of each ERAD component is described in the text.
Glucose
Mannose
GlucNAc
Glc3Man9GlcNAc2-unfolded protein
glucosidase I, II
CNX / CRT
glucosidase II
UDP-GP
Glc1Man9GlcNAc2-unfolded protein
Man9GlcNAc2-unfolded protein
Man9GlcNAc2-folded protein
Man8GlcNAc2-folded protein
Glc1Man8GlcNAc2-unfolded protein Man8GlcNAc2-unfolded protein
ERAD
Golgi apparatus
ER
EDEM
mannosidase I
mannosidase I
Fig. 2. Folding and degradation of glycoprotein. Sugar chains of nascent glycoproteins synthesized in the ER are trimmed by glucosidase I or
II, and polypeptides containing one glucose residue are folded by the calnexin cycle. One mannose residue of polypeptides that is unable to
be folded by the calnexin cycle is removed by mannosidase I, and then the polypeptides are recognized by EDEM and degraded by ERAD.
H. Yoshida ER stress and diseases
FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 633
domain, which may be responsible for recognition of
mannose residues.
Osteosarcoma 9 (OS9) and XTP3-transactivated
gene B (XTP3B) are other ERAD components respon-

sible for the recognition of unfolded proteins [26–28].
OS9 specifically binds to unfolded glycoproteins con-
taining eight (or five) mannose residues. OS9 also
binds to unglycosylated unfolded proteins, suggesting
that it plays a critical role in the recognition of both
glycosylated and unglycosylated proteins. OS9 and
XTP3B [29] contain the mannose-6-phosphate recep-
tor-like domain, which may be critical to the recogni-
tion of mannose residues.
Retrotranslocation
Nascent glycoproteins recognized by EDEM and OS9
as malfolded are destined for the retrotranslocation
machinery [30,31]. Before their retrotranslocation, nas-
cent proteins associate with PDI and BiP to cleave
disulfide bonds and to unfold the partially folded struc-
ture, respectively [32–34]. Although unfolded ER pro-
teins were previously speculated to be retrotranslocated
through the translocon containing Sec61, the molecular
structure of the retrotranslocation machinery remains
elusive. Derlin-1 is a mammalian homolog of yeast
Der1, and thought to be a critical component of the
machinery. Derlin-1 may form a retrotranslocation
channel in the ER membrane and associates with p97
through an adaptor protein, valocin-containing
protein (VCP)-interacting membrane protein 1
(VIMP1) [35]. Derlin-2 and Derlin-3, other Der1 homo-
logs, are also involved in ERAD [35–37], although the
exact underlying mechanism is still unclear.
p97 ⁄ cdc48 ⁄ VCP is a cytosolic AAA-ATPase and
recruits unfolded ER proteins to the cytosol [38,39].

Ubiquitin fusion degradation protein 1 (Ufd1) and
nuclear protein localization 4 (Npl4) bind to p97 as a
cofactor and help p97 to extract unfolded proteins.
The polypeptide portion of unfolded proteins interacts
with p97, whereas the polyubiquitin chains attached to
them are recognized by both p97 and Ufd1 and may
activate the ATPase activity of p97 [40–42].
Ubiquitination
Retrotranslocated (or retrotranslocating) proteins are
ubiquitinated by the E1–E2–E3 ubiquitin system.
Ubiquitin is first conjugated to enzyme E2 by enzyme
E1, and then transferred to ERAD substrates by
enzyme E3. HMG-CoA reductase degradation pro-
tein 1 (HRD1), gp78, and TEB4 ⁄ Doa10 are mem-
brane-anchored E3 ligases involved in ERAD [43–46],
whereas ubiquitin conjugase (UBC)6 and UBE2-
G2 ⁄ UBC7 are E2 conjugase involved in ERAD. UBE1
is an E1 ubiquitin-activating enzyme that is ubiqui-
tously involved in protein degradation by the protea-
some. HRD1 shows a preference for substrates that
contain misfolded luminal domains, whereas Doa10
prefers transmembrane proteins containing misfolded
cytosolic domains (Doa10 also ubiquitinates cytosolic
proteins). These two distinctive ERAD systems are
called ERAD-L (luminal ERAD) and ERAD-C (cyto-
solic ERAD) [47,48]. EDEM and OS9 are thought to
specifically recognize ERAD-L substrates. Actually,
they form distinct ubiquitin–ligase complexes: the
HRD1 complex contains HRD1, OS9, HRD3, Derlin-
1, USA1, UBX2 and p97, whereas the Doa10 complex

consists of Doa10, UBX2 and p97 [49–51]. Substrates
containing misfolded transmembrane domains skip the
interaction to OS9 and HRD3, and directly associate
with the HRD1 complex, which is called the ERAD-M
pathway [49].
However, there are a lot of other E3 ligases involved
in the ERAD, and they preferentially recognize distinct
ERAD substrates. FBX2 (F-box only protein 2) is
another E3 ligase that specifically recognizes N-glycos-
ylated proteins located in the cytosol [52,53]. Parkin is
an E3 involved in Parkinson’s disease (see below). In
the case of cystic fibrosis transmembrane conductance
regulator, its folding status is sequentially monitored
by the two E3 ligase complexes, such as the RMA1
complex and the CHIP (C-terminus of Hsc70-interact-
ing protein) complex [54].
Molecules other than E1–E2–E3 enzymes are also
involved in ubiquitination. UBX2 binds to both p97
and E3 ligases such as HRD1 and Doa10 to recruit
E3 to p97 [55], whereas gp78 directly associates with
p97 [56]. The ubiquitin-domain protein, Herp (homo-
cysteine-induced endoplasmic reticulum protein),
associates with a complex containing HRD1, p97,
Derlin-1, and VCP-interacting membrane pro-
tein [57,58].
Degradation
Retrotranslocated and ubiquitinated proteins are
deglycosylated by peptide–N-glycanase before their
degradation by the proteasome, because bulky glycan
chains may hamper the entrance of substrates into

the proteasome pore. As peptide–N-glycanase is asso-
ciated with Derlin-1, it is possible that deglycosylation
occurs coretrotranslocationally [59]. Deglycosylated
substrates are then delivered to the proteasome. Dsk2
and Rad23 facilitate this delivery of ERAD substrates
[60].
ER stress and diseases H. Yoshida
634 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS
Response pathways for ER stress
The mammalian ER stress response has four mecha-
nisms: (1) translational attenuation; the enhanced
expression of (2) ER chaperones and (3) ERAD
components; (4) induction of apoptosis. These four
responses are regulated by the regulatory pathways as
described below (Fig. 4).
PERK pathway
PERK is a type I transmembrane protein located in the
ER, which senses the accumulation of unfolded pro-
teins in the ER lumen [61–63]. The luminal portion of
PERK is involved in sensing unfolded proteins,
whereas the cytoplasmic portion contains a kinase
domain. In the absence of ER stress, BiP binds to the
luminal domain of PERK and keeps it from being acti-
vated (Figs 4 and 5A). In response to ER stress, BiP is
released from PERK, and PERK is activated through
oligomerization and trans-phosphorylation [64]. Activa-
ted PERK phosphorylates and inactivates the a-subunit
of eukaryotic translational initiation factor 2 (eIF2a),
leading to translational attenuation. The phosphoryla-
tion of PERK is transient as the protein is dephosphor-

ylated by specific phosphatases such as CReP
(constitutive repressor of eIF2a phosphorylation), pro-
tein phosphatase 2C-GADD34, and p58IPK. CReP is
constitutively expressed, whereas the expression of
GADD34 and p58IPK is induced on ER stress by
PERK and activating transcription factor (ATF)6
pathways, respectively.
Interestingly, translation of the transcription factor
ATF4 is up-regulated by eIF2a-mediated translational
attenuation. There are several small ORFs in the
5¢-UTR of ATF4 mRNA (Fig. 5B). The ribosome first
binds to a 5¢-cap structure, slides on the ATF4
mRNA, and then starts translation at the small ORFs
with unphosphorylated (active) eIF2a. As the ribosome
is released from the ATF4 mRNA upon the termin-
ation of translation at the stop codon of small ORFs,
the ATF4 ORF cannot be translated in the absence of
ER stress. In contrast, as phosphorylated (inactive)
eIF2a cannot start translation, the probability that the
ribosome reaches the ATF4 ORF is increased in the
presence of ER stress. Thus, the translation of ATF4
ER
ER stresss
nucleus
AARE ERSE UPRE
4
SS
66
ER chaperone
ERAD component

CHOP
anti-oxidative stress
translation
6
GA
6 6
6
S2P S1P
ATF6 PERK
pATF6(N)
P
eIF2α
GADD34
p58IPK
CReP
translational
attenuation
?
4 ATF4
IRE1α
S U
DBD
AD
XBP1 pre-mRNA
DBD-AD
mature mRNA
NF-Y
pXBP1(S)
pXBP1(U)
Fig. 4. Mammalian response pathways for ER stress. Three response pathways (PERK, ATF6, and IRE1 pathways) regulate the mammalian

ER stress response. PERK, a transmembrane kinase, phosphorylates eIF2a to attenuate translation, and to up-regulate expression of ATF4,
leading to enhanced transcription of target genes such as CHOP. ATF6, a transmembrane transcription factor, is translocated to the Golgi
apparatus and cleaved by proteases such as S1P and S2P, leading to enhanced transcription of ER chaperone genes. IRE1, a transmem-
brane RNase, splices XBP1 pre-mRNA, and pXBP1(S) translated from mature XBP1 mRNA activates transcription of ERAD component
genes.
H. Yoshida ER stress and diseases
FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 635
is remarkably enhanced in response to ER stress. The
targets of ATF4 include CHOP (C ⁄ EBP homology
protein), a transcription factor involved in the induc-
tion of apoptosis, and proteins involved in amino-acid
metabolism such as asparagine synthetase or those
involved in resistance to oxidative stress [65].
eIF2a is also phosphorylated by other kinases, such
as dsRNA-dependent protein kinase (PKR), GCN2
(general control of amino-acid synthesis 2) and heme-
regulated translational inhibitor. These kinases are
activated by viral infections, amino-acid starvation,
and heme deficiency, respectively, indicating that trans-
lational attenuation and ATF4 induction is induced by
not only ER stress but also these physiological situa-
tions. Thus, the cellular response mediated by the
phosphorylation of eIF2a is called the integrated stress
response and is essential for cell survival [66].
ATF6 pathway
There is another sensor molecule, ATF6, on the ER
membrane [67–70]. ATF6 is a type II transmembrane
protein, the luminal domain of which is responsible for
the sensing of unfolded proteins. The cytoplasmic
portion of ATF6 has a DNA-binding domain con-

taining the basic-leucine zipper motif (bZIP) and a
PERK
IRE1
BiP
BiP
BiP
BiP
phosphorylation oligomerization
A
ER
ATF6
GLS
BiP
GLS
BiP
GLS
translocation to Golgi
B
ATF4 mRNA
ATF4 coding region
small ORFs
CAP
- ER stress
ATF4 mRNA
CAP
eIF2α
small peptide
+ ER stress
ATF4 mRNA
CAP

phosphorylated eIF2α
ATF4 protein
ribosome
eIF2α
Fig. 5. Activation of the PERK pathway. (A)
Activation of PERK, IRE1, and ATF6. In the
absence of ER stress, BiP prevents PERK,
IRE1, and ATF6 from being activated by
binding to these sensors. BiP prevents the
activation of IRE1 and PERK by keeping
them from being oligomerized, whereas BiP
inhibits the translocation of ATF6 by mask-
ing the Golgi-localization signal (GLS). When
BiP is sequestered from sensors by unfol-
ded proteins, these sensor molecules are
activated. (B) Regulation of ATF4 expres-
sion. In the absence of ER stress, most of
the eIF2a is active (not phosphorylated), and
translation starts at the small ORFs, leading
to the release of ribosomes before they
reach the ATF4 ORF. Upon ER stress, most
of the eIF2a becomes inactive (phosphoryl-
ated), and translation rarely starts at the
small ORFs, thus ribosomes can reach the
ATF4 ORF and induce translation of ATF4
protein.
ER stress and diseases H. Yoshida
636 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS
transcriptional activation domain. In the absence of
ER stress, BiP binds to the luminal domain of ATF6

and hinders the Golgi-localization signal, leading to
inhibition of ATF6 translocation (Fig. 5A) [71–75]. In
response to the accumulation of unfolded proteins, BiP
dissociates from ATF6, and ATF6 is moved to the
Golgi apparatus by vesicular transport (Fig. 4). In the
Golgi apparatus, ATF6 is sequentially cleaved by a
pair of processing proteases called site 1 protease (S1P)
and site 2 protease (S2P), and the resultant cytoplas-
mic portion of ATF6 [pATF6(N)] translocates into the
nucleus. In the nucleus, pATF6(N) binds to a cis-act-
ing element, the ER stress response element (ERSE),
and activates the transcription of ER chaperone genes
such as BiP, GRP94 and calreticulin [68]. The consen-
sus sequence of the ER stress response element is
CCAAT-(N9)-CCACG, and ATF6 binds to the
CCACG portion, whereas a general transcription fac-
tor, NF-Y (nuclear factor Y), binds to the CCAAT
portion.
The cleavage of ATF6 is unique, especially as the
second cleavage by S2P occurs in the transmembrane
region [75]. This process is called regulated intramem-
brane proteolysis (RIP), which is well conserved from
bacteria to mammals (Fig. 6). The most characterized
substrate of RIP is sterol response element-binding
protein (SREBP) [75]. SREBP is a transcription factor
that is located in the ER membrane like ATF6. Upon a
deficiency of sterol, SREBP is transported to the Golgi
apparatus, cleaved by S1P and S2P, and activates the
transcription of genes involved in the biosynthesis of
sterol. Thus, the activation of ATF6 and SREBP is

mainly regulated at the level of vesicular transport. The
regulation of the transport of SREBP has been well
characterized, and regulatory components such as the
sensor-escort protein SCAP (SREBP cleavage-activa-
ting protein) and the anchor protein INSIG (insulin-
induced gene 1) have been identified [76].
There are two genes for ATF6, called ATF6a and
ATF6b, which have a similar function and are ubiqui-
tously expressed [68,77]. Recently, several bZIP tran-
scription factors located in the ER and regulated by
RIP have been reported. cAMP response element-bind-
ing protein H (CREBH) is specifically expressed in
liver, and processed by S1P and S2P in response to
ER stress [78]. CREBH activates the transcription of
acute-phase response genes involved in acute inflam-
matory responses. OASIS (old astrocyte specifically
induced substance) is also cleaved by S1P and S2P in
response to ER stress in astrocytes and activates the
transcription of BiP [79]. A spermatid-specific tran-
scription factor, Tisp40 (transcript induced in spermio-
genesis 40), is also severed by S1p and S2P and
activates the transcription of EDEM [80]. These tissue-
specific ATF6-like molecules may contribute to the ER
stress response.
Fig. 6. Molecules regulated by RIP. RIP is conserved from bacteria to mammals, and is involved in various biological processes. SREBP sen-
ses a sterol deficiency and activates the transcription of genes involved in sterol synthesis. Cleavage of APP by RIP results in the production
of antibody, which is responsible for the onset of Alzheimer’s disease. Notch is a cell surface protein that is cleaved by RIP upon binding
Delta, leading to the activation of target genes involved in differentiation. Bacterial RseA protein anchors a transcription factor, r
E
, to keep it

inactive. In response to accumulation of unfolded proteins in the periplasm, RseA is cleaved by RIP, leading to transcriptional activation of
periplasmic chaperones.
H. Yoshida ER stress and diseases
FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 637
Luman ⁄ LZIP ⁄ CREB3 can be cut by S1P and S2P
and activates the transcription of EDEM through a
cis-acting element, unfolded protein response element
(UPRE), although ER stress cannot induce Luman
RIP [80–82]. CREB4 is transported to the Golgi
apparatus in response to ER stress, is cleaved by S1P
and S2P, and activates the transcription of BiP,
although cleavage is not observed upon ER stress [83].
These ATF6-like molecules, which are insensitive to
ER stress, might be activated in situations other than
ER stress and activate transcription of ER chaperones.
IRE1 pathway
The third sensor molecule in the ER membrane is
IRE1 (inositol requirement 1) [84–86]. The luminal
domain of IRE1 is similar to that of PERK and
involved in the sensing of unfolded proteins, whereas
the cytoplasmic domain contains a kinase domain and
an RNase domain. There are two genes for IRE1,
IRE1a and IRE1b. Upon ER stress, BiP suppression
of IRE1 activation is released, and IRE1 is activated
through dimerization and transphosphorylation (Figs 4
and 5A) [64]. Activated IRE1a converts XBP1 (x-box
binding protein 1) pre-mRNA into mature mRNA by
an unconventional splicing mechanism [69,87]. As the
DNA-binding domain and the activation domain are
encoded in ORFs in XBP1 pre-mRNA, a pro-

tein translated from pre-mRNA [pXBP1(U)] cannot
activate transcription. In contrast, a protein translated
from mature mRNA [pXBP1(S)] activates the tran-
scription of ERAD component genes such as EDEM,
HRD1, Derlin-2, and Derlin-3 through a cis-acting ele-
ment, unfolded protein response element, as these two
ORFs are joined in mature mRNA [37,88,89].
pXBP1(S) also induces the expression of proteins
involved in lipid synthesis and ER biogenesis, as well
as the expression of ER chaperones such as BiP,
p58IPK, ERdj4, PDI-P5 and HEDJ [90,91]. Thus,
XBP1 is essential to the function of cells that produce
large amounts of secretory proteins such as pancreatic
b-cells, hepatocytes, and antibody-producing plasma
cells [92–95].
The splicing of XBP1 pre-mRNA by IRE1a is quite
different from conventional mRNA splicing (Fig. 7A)
[69]. Conventional splicing is catalyzed by the spliceo-
some, and the consensus sequence at the exon–intron
border is GU-AG or AU-AC (Chambon’s rule). The
splicing reaction is sequential: the 5¢ site is cleaved first,
then the 3¢ site after a lariat structure is formed. In con-
trast, unconventional splicing of XBP1 pre-mRNA is
catalyzed by IRE1a and RNA ligase, and there is a pair
of stem–loop structures at the exon–intron border
instead of GU-AG or AU-AC. Moreover, the splicing
reaction is not sequential but random.
The most important difference between conventional
and unconventional splicing is where the reaction
occurs (Fig. 7B). Conventional splicing (nuclear spli-

cing) takes place in the nucleus, whereas unconven-
tional splicing (cytoplasmic splicing) occurs in the
cytoplasm. The biological significance of cytoplasmic
splicing is that pre-mRNA used for translation in the
cytoplasm can be spliced when it is necessary to
change the nature of the protein translated from the
mRNA, in response to extracellular or intracellular
signaling. In contrast, as nuclear splicing cannot splice
mRNA exported to the cytoplasm, it is necessary for
pre-mRNA to be transcribed de novo and spliced.
Thus, cytoplasmic splicing would be a very rapid, ver-
satile, and energy-efficient mechanism with minimal
waste as compared with conventional mRNA splicing.
Recently, it was found that pXBP1(U) encoded in
XBP1 pre-mRNA is a negative feedback regulator of
pXBP1(S). Thus, in the case of XBP1, pre-mRNA and
mature mRNA encode negative and positive regula-
tors, respectively, and their expression is switched by
cytoplasmic splicing in response to the situation in the
ER [96].
IRE1b is specifically expressed in epithelial cells
of the gastrointestinal tract, and thought to cleave
rRNA to attenuate translation in response to ER
stress [84]. When IRE1 b– ⁄ – mice were exposed to an
inducer of inflammatory bowel disease, they actually
developed colitis, possibly because of the enhanced
ER stress [97].
Recently, the crystal structure of the luminal domain
of IRE1a was solved [98]. The luminal domain is sim-
ilar in structure to the peptide-binding domain of

major histocompatibility complexes, suggesting the
interesting possibility that it directly senses ER stress
by directly binding unfolded proteins.
Apoptosis-inducing pathways
The accumulation of unfolded proteins in the ER is
toxic to cells. Thus, if the PERK, ATF6, and IRE1
pathways cannot suppress ER stress, an apoptotic
pathway is triggered to ensure survival of the organism
as a last line of defense. A number of pathways have
been reported to be involved in ER stress-induced
apoptosis, and the full induction of apoptosis seems to
require the concomitant activation of several death
pathways, although there remain many arguments over
ER stress-induced apoptosis [99–105]. In this section, I
will briefly summarize the known death pathways,
focusing on recent progress (Fig. 8).
ER stress and diseases H. Yoshida
638 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS
The most characterized pathway is the CHOP path-
way. CHOP ⁄ GADD153 (growth arrest and DNA
damage 153) is a transcription factor, the expression of
which is induced by the ATF6 and PERK pathways
upon ER stress [70,106,107]. CHOP– ⁄ – cells exhibit
less programmed cell death when faced with ER stress
[108], suggesting that the CHOP pathway is a major
regulator of ER stress-induced apoptosis. As for the
target genes of CHOP, CHOP activates the transcrip-
tion of GADD34, ERO1, DR5 (death receptor 5), and
carbonic anhydrase VI, which seem to be responsible
for apoptosis. GADD34 associated with protein phos-

phatase 2C enhances dephosphorylation of eIF2a and
promotes ER client protein biosynthesis [109], whereas
ERO1, which encodes an ER oxidase, makes the ER a
more hyper-oxidizing environment [110]. DR5, which
encodes a cell surface death receptor, may activate
caspase cascades [111]. Carbonic anhydrase VI may
change the cellular pH, affecting various cellular pro-
cesses [112,113]. However, the exact signaling mechan-
ism from CHOP to apoptosis is still unclear.
The second apoptotic pathway is the IRE1–TRAF2–
ASK1 pathway. The cytoplasmic part of IRE1 binds
to an adaptor protein, TRAF2 (tumor necrosis factor
receptor-associated factor 2), which couples plasma
membrane death receptor to Jun kinase (JNK) and
stress-activated protein kinase (SAPK) [114]. IRE1 and
TRAF2 form a complex with a mitogen-activated
protein kinase kinase kinase, ASK1 (apoptosis signal-
regulating kinase 1), and this IRE1–TRAF2–ASK1
complex is responsible for the phosphorylation and
activation of JNK [115]. Actually, IRE1– ⁄ – cells as
well as ASK1– ⁄ – cells are impaired in the activation
of JNK and apoptosis by ER stress. In contrast,
A
B
Fig. 7. Cytoplasmic splicing. (A) Comparison
between nuclear and cytoplasmic splicing.
Conventional splicing is catalyzed by the
spliceosome in the nucleus, and there is a
consensus sequence at the exon–intron
boundary such as GU-AG or AU-AC. The

splicing reaction is sequential: the 5¢ site is
cleaved first, the lariat structure is formed,
and then the 3¢ site is cleaved. In contrast,
unconventional splicing is catalyzed by IRE1
and RNA ligase in the cytoplasm, there is a
characteristic stem–loop structure at the
boundary, and the splicing reaction is ran-
dom without forming a lariat structure. (B)
Biological significance of cytoplasmic spli-
cing. As nuclear splicing cannot splice pre-
mRNA exported to the cytoplasm, de novo
transcription is required to change the char-
acter of the protein encoded in the pre-
mRNA. In contrast, as cytoplasmic splicing
can splice pre-mRNA that is translated in
the cytoplasm, it can rapidly change the
character of a protein in response to exter-
nal or internal stimuli, without de novo
transcription.
H. Yoshida ER stress and diseases
FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 639
TRAF2– ⁄ – cells are more susceptible to apoptosis trig-
gered by ER stress, which might be inconsistent with
the above model [116]. TRAF2 also associates with
caspase-12 and regulates its activation [117]. IRE1–
TRAF2 activates the transcriptional repressor ATF3
as well, leading to the activation of apoptosis [118].
These suggest that the IRE1–TRAF2–ASK1 pathway
is a major regulator of ER stress-induced apoptosis.
TNFR1 (tumor necrosis factor receptor 1), a receptor

for TNF-induced cell death, associates with IRE1a
upon ER stress, and the activation of JNK by ER
stress is impaired in TNFR1– ⁄ – cells. This suggests
that TNFR1 mediates the ER stress-induced activation
of JNK [119], possibly forming a complex with IRE1a,
TRAF2, and ASK1. The expression of TNFa is
up-regulated by the IRE1 pathway during ER stress
[120,121], which may contribute to the activation of
TNFR1.
Caspases are well-known pro-apoptotic components,
and caspases 2, 3, 4, 7, 9 and 12 are reported to be
involved in ER stress-induced cell death [122–131].
Caspase-12 is associated with the ER membrane, and
activated by ER stress, possibly by calpain [132]. Then
caspase-12 activates caspase-9, which in turn activates
caspase-3 [133], leading to cell death. Caspase-12– ⁄ –
mice are resistant to ER stress-induced apoptosis
but sensitive to other death stimuli, suggesting that
caspase-12 is a regulator specific to ER stress-induced
apoptosis [134]. The activation of caspase-12 by ER
stress is observed in models of various diseases such as
Alzheimer’s disease, polyglutamine disease, ischemia,
and viral infection [135–139], suggesting that ER
stress-induced apoptosis is closely involved in these
diseases (see below). However, the involvement of
caspase-12 in apoptosis of human cells is still open to
question, as the human caspase-12 gene contains sev-
eral mutations critical to function [140]. It is possible
that an unidentified caspase other than caspase-12 is
responsible in human cells.

Bcl2 family proteins are well-known components of
the programmed cell death machinery, and some key
components are involved in ER stress-induced apopto-
sis. In general, pro-apoptotic members of the Bcl2
family seem to be recruited to the ER surface and
to activate caspase-12, whereas the anti-apoptotic
members inhibit this recruitment, although the exact
Fig. 8. ER stress-induced apoptotic pathways. The subcelluar distribution of factors involved in ER stress-induced cell death is shown. Pro-
apoptotic and anti-apoptotic factors are indicated in black and white letters, respectively. Pro-apoptotic Bcl2 proteins positively regulate the
IRE1 and caspase pathways, whereas anti-apoptotic Bcl2 proteins negatively regulate the latter. p53 enhances expression of pro-apoptotic
Blc2 family proteins such as PUMA and NOXA. The IRE1 pathway activates JNK and SAPK, leading to apoptosis. c-Abl translocates from
the ER to the mitochondria in response to ER stress. A fuller explanation is given in the text.
ER stress and diseases H. Yoshida
640 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS
relationship between these factors is still unclear. So I
will briefly describe our current understanding of Bcl2
family proteins. The anti-apoptotic factor Bcl2 is
down-regulated by the transcription factor CHOP
upon ER stress, which leads to enhanced oxidant
injury and apoptosis [141]. Overexpression of Bcl2
inhibits the activation of caspase-12 and apoptosis
during ER stress [142]. The pro-apoptotic factor, BAD
(Bcl2 antagonist of cell death), is dephosphorylated
and activated in response to ER stress [143], whereas
other pro-apoptotic factors, Bax (Bcl2-associated X
protein) and Bak (Bcl-2 homologous antagonist ⁄ kil-
ler), are present in the ER membrane as well as the
mitochondrial membrane [144,145]. During ER stress,
Bax and Bak oligomerize and activate caspase-12.
Interestingly, Bax and Bak associate with IRE1a and

modulate IRE1a function during ER stress [146]. Bax
and Bak are required for most forms of apoptosis
[147]. The transcription of PUMA (p53 up-regulated
modulator of apoptosis) and NOXA (neutrophil
NADPH oxidase factor), pro-apoptotic members of
the BH3 (homology domain-3) domain-only family, is
up-regulated by p53 during ER stress, and PUMA– ⁄ –
cells and NOXA– ⁄ – cells are resistant to ER stress-
induced apoptosis [148,149]. Another pro-apoptotic
component, Bim (Bcl2-interacting mediator of cell
death), translocates from the dynein-rich compartment
to the ER membrane and activates caspase-12 in
response to ER stress, whereas an anti-apoptotic fac-
tor, Bcl-xL (Bcl-2-like 1), binds to Bim and inhibits its
translocation [150]. Bim-knockdown cells are resistant
to ER stress. The ER-localized anti-apoptotic factor
BI-1 (Bax inhibitor-1) inhibits the activation of Bax
during ER stress, and BI-1– ⁄ – mice are sensitive to
ER stress, whereas mice overexpressing BI-1 are resist-
ant [151]. BIK (Bcl2-interacting killer) is an ER-locali-
zed pro-apoptotic component which enhances the
recruitment of BAX and BAK to the ER [152]. Bap31
(B cell receptor-associated protein 31) is a pro-apop-
totic factor that is cleaved and activated upon ER
stress, its cleavage being dependent on calnexin [153].
In calnexin-deficient cells, the cleavage of Bap31 and
ER stress-induced apoptosis are inhibited.
The inhibitor of apoptosis (IAP) family has also
been reported to be involved in ER stress-induced
apoptosis. Human inhibitor of apoptosis 2 (HIAP2) is

an IAP that inhibits caspase-3 and caspase-7. Expres-
sion of HIAP2 is induced upon ER stress at the level
of translation: caspases activated by ER stress cleave
eukaryotic initiation factor, p97 ⁄ DAP5 ⁄ NAT1, and
the cleavage product specifically activates the HIAP2
internal ribosome entry site, leading to enhanced trans-
lation of HIAP2 [131]. Transcription of IAP-2 and
XIAP (inhibitor of apoptosis, X-linked), two other
IAPs, is up-regulated during ER stress, and cells in
which these IAPs have been knocked down are sensi-
tive to ER stress-induced apoptosis [154]. Cells overex-
pressing XIAP or HIAP1 are resistant to ER stress
[122,155]. These results suggest involvement of IAP
proteins in ER stress-induced apoptosis.
c-Abl (Abelson murine leukemia viral oncogene
homolog 1) is a protein tyrosine kinase distributed in
the nucleus and cytoplasm, and c-Abl activated by a
death signal induces phosphorylation and activation of
pro-apoptotic JNK and SAPK. Interestingly, c-Abl is
also located on the ER surface, and translocates to the
mitochondria upon ER stress, where it induces the
release of cytochrome c [156]. c-Abl– ⁄ – cells are resist-
ant to ER stress-induced apoptosis. A c-Abl-interact-
ing protein, Aph2 (anterior phalynx defective 2), is
also located on the ER and shows pro-apoptotic acti-
vity [157], suggesting that c-Abl forms a distinct path-
way leading to ER stress-induced apoptosis.
PKR is a dsRNA-dependent protein kinase which is
activated upon viral infection, and its phosphorylation
in the nucleus is up-regulated in response to ER stress.

Interestingly, PKR-knockdown cells or PKR mutant
cells are resistant to ER stress-induced apoptosis, sug-
gesting that PKR is a pro-apoptotic factor during ER
stress [158].
TDAG51 (T-cell death-associated gene 51) is a mem-
ber of the pleckstrin homology-related domain family,
and its transcription is induced by ER stress through
the PERK pathway [159]. Overexpression of TDAG51
induces apoptosis, suggesting that TDAG51 is involved
in ER stress-induced apoptosis and the development of
atherosclerosis (see below).
Nuclear respiratory factor (NRF)1 and NRF2 are
transcription factors that regulate the oxidative stress
response. NRF2 is distributed in the cytoplasm
through its association with the microtubule-associated
protein Keap1 (Kelch-like Ech-associated protein 1).
Upon ER stress, PERK phosphorylates NRF2 and
dissociates it from Keap1, leading to the nuclear
recruitment of NRF2 [160]. Remarkably, NRF2– ⁄ –
cells are sensitive to ER stress-induced apoptosis,
whereas NRF1 is located in the ER membrane, and
translocates to the nucleus upon ER stress [161], sug-
gesting that these proteins are involved in ER stress-
specific apoptosis.
ATF6 is also involved in the apoptotic process dur-
ing myogenesis. In differentiating myoblast, the ATF6
pathway is activated, and expression of BiP and CHOP
is up-regulated, which may activate caspase-12. More-
over, AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride
hydrochloride], an inhibitor of ATF6 activation, blocks

H. Yoshida ER stress and diseases
FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 641
apoptosis, suggesting that the ATF6 pathway contri-
butes to apoptosis during myogenesis [162].
TRB3 (Tribbles homolog 3) is a human ortholog of
Drosophila tribble, and its transcription is induced by
ER stress through the PERK–ATF4–CHOP pathway.
Interestingly, TRB3-knockdown cells are resistant to
ER stress-induced apoptosis, suggesting that TRB3 is
a pro-apoptotic factor during ER stress [163,164].
p53 is a transcription factor that induces growth
arrest and apoptosis in response to various forms of
cellular stress, such as DNA damage. There are several
reports suggesting a connection between p53 and ER
stress. Upon ER stress, p53 is phosphorylated by gly-
cogen synthase kinase 3B, leading to the distribution
of p53 in the cytoplasm and destabilization of p53,
and attenuation of p53-dependent apoptosis [165–167].
Interestingly, Scotin and SCN3B (sodium channel sub-
unit beta 3), p53-inducible pro-apoptotic proteins, are
located in the ER [168,169]. Moreover, transcription of
PUMA and NOXA, which are involved in ER stress-
induced apoptosis, is up-regulated by p53 during ER
stress [148], suggesting that the p53 pathway regulates
the apoptotic pathway during ER stress.
Other components such as VCP and ALG-2 (apop-
tosis-linked-gene 2) [104], AIGP1 [170], elongation
factor-1a [171], and NRADD (neurotrophin receptor
associated death domain) [172] are also reported to be
involved in ER stress-induced apoptosis. Their precise

functions and working mechanisms are still to be clar-
ified.
ER stress-related diseases
Unfolded or malfolded proteins readily form aggre-
gates in the ER as well as the cytosol. Recent reports
suggested that small aggregates are highly toxic, as
they impair the ubiquitin proteasome pathway [173]
and sequester transcription factors such as CREB-
binding protein and TATA-binding protein [174,175],
whereas large aggregates such as the aggresome and
the inclusion body are cytoprotective [176]. Interest-
ingly, molecular chaperones such as HSP70 and TRiC
(TCP1- ring complex) can suppress the formation as
well as toxicity of protein aggregates [174,177–181].
The diseases caused by the malfolding of cellular pro-
teins are collectively called ‘conformational diseases’ or
‘folding diseases’. As malfolded proteins and protein
aggregates can evoke ER stress, it has been speculated
that ER stress is involved in most conformational dis-
eases, particularly Alzheimer’s disease and Parkinson’s
disease, although it is still heavily disputed whether
ER stress (or protein aggregates) is a major cause of
these diseases. In this section, I will briefly summarize
recent findings on how ER stress is involved in con-
formational diseases.
Neurodegenerative diseases
Neurons are thought to be sensitive to protein aggre-
gates, and there are many reports that ER stress is
involved in neurodegenerative diseases [182–184]. In
fact, disruption of SIL1⁄ BAP, a cochaperone of BiP,

results in the accumulation of protein aggregates and
neurodegeneration [184]. Most of these diseases are
caused by aging or genetic background, although
several are assumed to be infectious, especially prion
disease.
Alzheimer’s disease is the most common neurode-
generative disease, and characterized by cerebral neu-
ritic plaques of amyloid b-peptide [185–191]. Recent
findings strongly suggest that one of the major causes
of Alzheimer’s disease is an accumulation of amyloid
b-peptide, although tau also seems to be involved in
the disease. Studies of patients with autosomal-domin-
ant familial Alzheimer’s disease have identified three
genes responsible for the disease, amyloid precursor
protein (APP), PS1 and PS2. The amyloid precursor
protein encoded by APP is a transmembrane protein,
the function of which is still unknown, whereas both
PS1 and PS2 encode a protein called presenilin, which
is an essential component of a protease called c-secret-
ase. APP is sequentially cleaved by a b-secretase called
BACE (b-site amyloid b A4 precursor protein-cleaving
enzyme 1) and c-secretase, leading to the accumulation
of amyloid b-peptide. Interestingly, cells expressing
PS1 mutants show a waned ER stress response and are
sensitive to ER stress [192]. The activation of ATF6,
IRE1, and PERK is also disturbed in these mutant
cells [193]. Moreover, proteins involved in ER stress-
induced apoptosis, such as PKR and caspase-4, are
involved in the onset of Alzheimer’s disease [125,158].
Actually, the ER stress response is activated in patients

with Alzheimer’s disease [192,194,195], and poly-
morphism of SEL1, a component of ERAD, is linked
to Alzheimer’s disease [196]. These findings strongly
suggest a strong causal relationship between ER stress
and Alzheimer’s disease, and it is highly possible that
ER stress invoked by the accumulation of amyloid
b-peptide is one of the key mechanisms of Alzheimer’s
disease.
Parkinson’s disease is the second most common
neurodegenerative disease, which is characterized by a
loss of dopaminergic neurons [197]. Analyses of patients
with familial Parkinson’s disease have revealed three
genes responsible for the disease, encoding a-synuc-
lein, Parkin, and ubiquitin C-terminal esterase L1
ER stress and diseases H. Yoshida
642 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS
(UCH-L1). a-synuclein is a cytoplasmic protein which
forms aggregates, called Lewy bodies, characteristic of
Parkinson’s disease, but the link between a-synuclein
and ER stress is unclear. In contrast, Parkin is a ubiqu-
itin-protein ligase (E3) involved in ERAD [198]. One of
the substrates of ERAD ubiquitinated by Parkin is Pael
receptor, a homolog of endothelin receptor type B [199].
Interestingly, expression of Parkin is induced by ER
stress, and neuronal cells overexpressing Parkin are
resistant to ER stress [200]. As for UCH-L1, it is an
abundant protein in neurons and stabilizes a monomeric
ubiquitin [201,202]. It has been shown that UCH-L1
ubiquitinates unfolded proteins and might be involved
in ERAD [203]. These findings strongly suggest the

involvement of ER stress in Parkinson’s disease. In
addition, there are several other reports supporting the
link between ER stress and Parkinson’s disease. First,
Parkinson’s disease mimetics, such as 6-hydroxydopam-
ine, specifically induce ER stress in neuronal cells
[204,205]. Second, expression of ER chaperones such as
PDI is up-regulated in the brain of Parkinson’s disease
patients, and PDI is accumulated in Lewy bodies
[206]. The incidence of sporadic Parkinson’s disease
increases with age, but it is still unclear whether the ER
stress response wanes in patients with Parkinson’s
disease.
Polyglutamine (polyQ) diseases are neurodegenera-
tive disorders caused by duplications of the CAG-
repeat in certain genes, and include Huntington’s
disease, spinobulbar muscular atrophy (Kennedy dis-
ease), Machado-Joseph disease, dentatorubral-pallido-
luysian atrophy (Haw River Syndrome), and
spinocerebellar ataxia. Large polyQ stretches translated
from the CAG-repeat form insoluble protein aggre-
gates, which are toxic to cells. Although the exact
mechanism behind the toxicity of polyQ remains to be
clarified, one possibility is that polyQ aggregates
sequester other normal proteins, such as transcription
factors, which are indispensable to cell function [174].
Another possibility is that the polyQ aggregate itself is
toxic to cells. All known polyQ proteins causing neu-
rodegenerative diseases are cytosolic, but they evoke
ER stress, as polyQ proteins suppress the function of
the proteasome, which is an essential component of

ERAD [115,136,207]. Interestingly, p97, a component
of the ERAD machinery, enhances degradation of po-
lyQ proteins and suppresses polyQ protein-induced
neurodegeneration [208]. Judging from these findings,
it is probable that ER stress is involved in the onset of
polyQ diseases.
Pelizaeus-Merzbacher disease is a progressive neuro-
degenerative disorder characterized by a loss of coordi-
nation, motor abilities, and intellectual function [209].
Currently, it is thought that the disease is caused by a
mutation of the PLP1 gene which encodes a transmem-
brane proteolipid essential for the maintenance of mye-
lin sheaths, as well as in oligodendrocyte development
and axonal survival [210,211]. As a missense mutation
causes a more severe phenotype than a null mutation,
it is speculated that the missense mutant of PLP1
forms aggregates in the ER and evokes ER stress-
induced cell death [212]. Actually, expression of
CHOP, BiP, ERp59, and ERp72 is up-regulated in the
brains of mice expressing missense PLP and the brains
of patients with Pelizaeus-Merzbacher disease.
Prion disease, also called transmissible spongi-
form encephalopathy, encompasses Creutzfeldt-Jakob
disease, Gerstmann–Straussler–Scheinker syndrome,
fatal familial insomnia, Kuru, Alpers syndrome,
bovine spongiform encephalopathy, transmissible milk
encephalopathy, chronic wasting disease, and scrapie
[213]. Characteristic features of the disease are a loss
of motor control and dementia. The only gene to be
identified so far as being responsible for prion dis-

ease is PrP, which encodes a protein anchored to the
cell surface. There is no difference in amino-acid
sequence between the normal protein PrP
c
and its
pathological form PrP
Sc
. PrP
Sc
is rich in b-sheets,
converts PrP
c
into PrP
Sc
, and forms amyloid fibrils
that are thought to be toxic to cells. Interestingly, in
murine cells infected with PrP
Sc
, the expression of
ER chaperones such as GRP58 and GRP94 as well
as caspase-12 is up-regulated [214]. Moreover, the
expression of these chaperones is considerably
increased in patients with Creutzfeldt-Jakob disease.
Finally, overexpression of GRP58 protects cells from
PrP
Sc
-induced cell death, whereas inhibition of
GRP58 expression with small interfering RNA results
in a severe phenotype [215]. These findings strongly
suggest that ER stress is involved in the pathology

of prion disease.
Amyotrophic lateral sclerosis, also called Lou Geh-
rig’s disease, is a progressive neuromuscular disease
and shows characteristic pathological features such as
a loss of motor neurons in the cerebral cortex and spi-
nal cord. Analysis of patients with familial amyotroph-
ic lateral sclerosis has revealed that superoxide
dismutase-1 is responsible for the disease. Mutant
superoxide dismutase-1 forms aggregates in the ER,
evokes ER stress, induces the expression of BiP, and
activates caspase-12, leading to neuronal cell death.
These findings support the notion that ER stress
induced by superoxide dismutase aggregates is a major
cause of amyotrophic lateral sclerosis [216–218].
GM1 gangliosidosis is an autosomal recessive
lysosomal storage disorder characterized by an
H. Yoshida ER stress and diseases
FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 643
accumulation of GM1 gangliosides in the brain. The
disease is caused by a mutation of lysosomal b-galac-
tosidase that converts GM1 gangliosidosides to GM2
gangliosidosides. The analysis of b-galactosidase-
knockout mice has revealed that the accumulation of
GM1 gangliosidosides evokes ER stress, induces the
expression of BiP and CHOP, activates JNK2 and
caspase-12, and causes the apoptosis of neurons [219].
These findings suggest a strong correlation between
ER stress and GM1 gangliosidosis.
Bipolar disorder
Bipolar disorder is a class of mood disorders where

the person experiences recurrent episodes of mania
and depression [220]. Genetic linkage studies suggest
that genetic factors are involved in the disease.
Microarray analyses of cells derived from twins dis-
cordant with respect to the disease revealed that the
expression of XBP1 and BiP wanes in the affected
twins. Recent reports showed that there are polymor-
phisms in the promoter regions of XBP1 and BiP
that are common to patients [221–224], although this
conclusion is still controversial [225]. It is interesting
that mood-stabilizing drugs, such as valproate and
lithium, which are highly effective in the treatment of
bipolar disorder can increase the expression of ER
chaperones such as BiP, GRP94, and calreticulin
[226]. Further analyses are required to confirm that
ER stress is involved in the onset of bipolar dis-
order.
Diabetes mellitus
Diabetes mellitus is a disease characterized by hyper-
glycemia caused by the impaired secretion or action of
insulin [227–229]. Type I diabetes mellitus [insulin-
dependent diabetes mellitus (IDDM)] results from
selective destruction of insulin-secreting b-cells,
whereas type II [noninsulin-dependent diabetes mellitus
(NIDDM)] is characterized clinically by insulin resist-
ance. As b-cells seem to suffer from ER stress caused
by the production of a large amount of insulin, the
link between ER stress and IDDM has been investi-
gated thoroughly, especially the PERK pathway.
PERK is responsible for an early infancy IDDM called

Wolcott–Rallison syndrome [230], and PERK– ⁄ – mice
show symptoms characteristic of IDDM [62,231].
Moreover, mice with a homozygous mutation at the
eIF2a phosphorylation site (Ser51Ala) show similar
pathological features [232,233], whereas the onset of
diabetes is delayed in CHOP– ⁄ – mice [234]. Interest-
ingly, components of ER stress pathways other than
the PERK pathway are also involved in IDDM. First,
the expression of BiP, GRP94, ORP150 and HRD1 is
up-regulated, and both ATF6 and XBP1 are activated
in the Akita diabetes mouse model [235,236]. Second,
p58IPK– ⁄ – mice also show symptoms resembling
IDDM [237]. Finally, the expression of WFS1, a gene
responsible for the onset of Wolfman syndrome
(juvenile diabetes), is induced by ER stress, and
knockdown or knockout of WFS1 causes ER stress in
pancreatic b-cells [238,239]. These findings strongly
suggest that ER stress is deeply involved in the onset
of IDDM.
There are several reports suggesting that ER stress
is also involved in NIDDM. First, obesity, one of the
causes of NIDDM, evokes ER stress, and XBP1– ⁄ –
mice develop insulin resistance [240], although the
underlying mechanism is unknown. Second, ectopic
expression of ORP150 in b-cells improves insulin
tolerance [240,241]. A possible explanation for this is
that ORP150 expression may improve the folding
capacity in the ER and protect cells from ER stress-
induced apoptosis. Finally, a small nuclear poly-
morphism analysis revealed that a polymorphism in

ATF6 is associated with NIDDM in Pima Indians
[242].
Atherosclerosis
Atherosclerosis is a disease wherein arteries harden
and narrow because of the accumulation of fatty sub-
stances, cholesterol, cellular waste, Ca
2+
, and other
substances in the arterial inner lining, leading to heart
attack or stroke. One of the risk factors for athero-
sclerosis is the accumulation of homocysteine, which
is an intermediate produced during the metabolism of
sulfur amino acids [243,244]. Interestingly, homocyste-
ine induces ER stress by an unknown mechanism,
and then increases the expression of BiP, GRP94,
CHOP, Herp, and TDAG51 [159,245,246]. The ER
stress induced by homocysteine also increases choles-
terol synthesis by activating the transcription factor
SREBP. Both the induction of CHOP and TDAG51
(a member of the pleckstrin homology-related
domain family) and the accumulation of cholesterol
appear to promote the apoptosis of macrophages,
leading to the deposition of macrophage debris in
blood vessels that contributes to the development of
the atherosclerosis observed in hyperhomocysteinemia
[247–249]. Actually, CHOP– ⁄ – macrophages are less
sensitive to cholesterol-induced apoptosis. These find-
ings strongly support the notion that ER stress-
induced apoptosis is one of the major factors that
cause atherosclerosis.

ER stress and diseases H. Yoshida
644 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS
Inflammation
Inflammation is the first response of the immune sys-
tem to infection. Although the mechanism of inflam-
mation is complicated, ER stress is involved in some
types of inflammation. In inflammation of the central
nervous system, interferon-c induces ER stress and
apoptosis of oligodendrocytes [250]. Interestingly,
PERK
+
mice show enhanced central nervous system
hypomyelination and oligodendrocyte loss, suggesting
that the PERK pathway has a protective role against
interferon-c-induced apoptosis. In the case of lipopoly-
saccharide-induced inflammation of the lungs, lipo-
polysaccharide induces ER stress and CHOP
expression, leading to the apoptosis of lung cells [251].
Diclofenac, a nonsteroidal anti-inflammatory drug,
suppresses ER stress-induced apoptosis [252], suggest-
ing that ER stress is one of the major mediators of
inflammation. Nitric oxide (NO) is another substance
involved in the induction of apoptosis during inflam-
mation. Although NO-induced apoptosis has been gen-
erally considered to be mediated by either DNA
damage or mitochondrial damage, NO also induces
apoptosis mediated by ER stress and CHOP in pancre-
atic b-cells, microglial cells, and macrophages [103,229,
234,253–257]. Recently, it was reported that proinflam-
matory cytokines and lipopolysaccharide evoke ER

stress and induce the transcription and activation of
CREBH in the liver [78]. The transcription factor
CREBH induces the expression of proteins involved in
the acute inflammatory response such as serum amy-
loid P-component and C-reactive protein. As men-
tioned above, CREBH is structurally related to ATF6
and activated by a mechanism similar to ATF6. These
findings suggest a strong link between ER stress and
inflammation, although it should be clarified how ER
stress is induced during inflammation.
ER stress is also involved in autoimmunity, and
there are three reports supporting this notion. First,
BiP associates with the clinically relevant autoantigen
Ro52 (ribonucleoprotein autoantigen 52 kDa) [Sjoe-
gren syndrome type A antigen (SS-A)], and is thought
to be involved in autoimmunity and rheumatoid arthri-
tis [258]. Second, a microarray analysis using muscle
tissue of patients with myositis revealed that the
expression of BiP, CHOP, GADD45 and asparagine
synthetase is induced in the patients’ cells, suggesting
that the ER stress response is responsible for the skel-
etal muscle damage and dysfunction in autoimmune
myositis [259]. Third, overexpression of synoviolin, an
E3 ubiquitin ligase involved in ERAD, in mice causes
arthropathy with synovial hyperplasia, whereas knock-
down of synoviolin results in increased apoptosis of
synovial cells and less sensitivity to collagen-induced
arthritis [260].
Ischemia
As hypoxia induces ER stress as mentioned above, ER

stress is an important cause of ischemia-related dis-
eases. For instance, brain ischemia induces ER stress
in neurons and activates the ATF6, IRE1 and PERK
pathways [261], leading to the CHOP-mediated apop-
tosis of neurons [262]. Ischemia also induces ER stress
and the expression of ER chaperones in the heart,
leading to degeneration of cardiomyocytes [263], sug-
gesting that ER stress is involved in the development
of ischemic heart disease (see below).
Heart diseases
It has been reported that ER stress is involved in heart-
related diseases. Pressure overload by transverse aortic
constriction induces expression of ER chaperones and
ER stress-induced apoptosis of cardiac myocytes, lead-
ing to cardiac hypertrophy [264]. Interestingly, trans-
genic mice expressing a mutant KDEL receptor, a
retrieval receptor for ER chaperones in the early secre-
tory pathway, developed dilated cardiomyopathy due
to a malfunction of the ER quality control machinery
and increased ER stress [265]. Moreover, up-regulation
of ER chaperones protected cardiomyocytes from ER
stress-induced apoptosis [266]. These findings strongly
suggest that the ER stress response is essential for
homeostasis of cardiomyocytes.
Liver diseases
Hepatocytes have a well-developed ER structure that is
essential for the vigorous synthesis of secretory pro-
teins, and it has been reported that ER stress is
involved in liver-related diseases [267]. For instance,
alcohol is known to cause liver injury by various mech-

anisms including ER stress [268,269]. Although exactly
how alcohol causes ER stress remains unclear; one
possible explanation is that alcohol inhibits the activity
of the key enzymes for sulfur amino-acid metabolism
such as methionine synthetase and betaine-homocyste-
ine methyltransferase, leading to the accumulation of a
potent ER stress inducer, homocysteine [121,270]. ER
stress evoked by alcohol activates transcription factors
such as CHOP, leading to CHOP-induced apoptosis of
hepatocytes. ER stress also activates another transcrip-
tion factor, SREBP, which is responsible for the
up-regulation of fatty acid synthesis, resulting in lipid-
induced apoptosis [268,271].
H. Yoshida ER stress and diseases
FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 645
ER stress is also involved in hepatocarcinogenesis
[272–274]. In human hepatocellular carcinoma, the
ATF6 and IRE1 pathways are activated, and expres-
sion of BiP is markedly increased, suggesting that the
transformation of hepatocytes induces ER stress, and
cells cope with the stress by activating the ER stress
response pathways.
Kidney diseases
Various factors are known to induce renal injury by
evoking ER stress. First, the analgesic and antipyretic
drug paracetamol (acetaminophen) causes renal tubu-
lar injury, and ER stress-induced apoptosis is one of
the mechanisms involved [275]. Actually, paracetamol
induces an ER stress response that includes the induc-
tion of CHOP and cleavage of caspase-12. Second,

complement attack also induces ER stress and acti-
vates the PERK pathway, leading to glomerular epi-
thelial cell injury [276]. Third, excessive accumulation
of secretory proteins induces podocyte injury by evo-
king ER stress [277]. Fourth, overexpression of a
kidney-specific serine protease inhibitor, megsin, indu-
ces ER stress and renal injury [278]. Interestingly, prior
induction of BiP expression protects renal epithelial
cells from chemicals inducing renal injury [279]. These
findings suggest that ER stress is one of the major cau-
ses of chemically induced renal injury, and that the ER
stress response is a defense mechanism against renal
injury.
Viral infection
Infections caused by various viruses such as hepatitis B
virus [280,281], hepatitis C virus [282], hepatitis D
virus [283], flavivirus [284], Borna disease virus [285],
murine leukemia virus [286] and Moloney murine
leukemia virus [127] are known to induce ER stress,
possibly because the vigorous synthesis of viral pro-
teins makes the ER very busy. Upon viral infection,
cells activate the ER stress response pathways to pro-
tect them from ER stress-induced apoptosis. ER stress
induced by a viral infection is thought to cause various
pathogenetic effects such as neurodegeneration, liver
injury and carcinogenesis, depending on the cell types
infected [267].
Hereditary tyrosinemia type I
Hereditary tyrosinemia type I is a metabolic disease
affecting mainly the liver and renal functions, which is

caused by a deficiency of fumarylacetate hydrolase
which catalyzes the hydrolysis of fumarylacetate. In
cells of patients with hereditary tyrosinemia type I,
fumarylacetate is accumulated and evokes ER stress as
well as mutagenic, cytostatic and apoptotic effects and
chromosomal instability [287]. In hamster lung cells,
fumarylacetate induces an ER stress response that
includes the induction of BiP and CHOP expression
and the cleavage of caspase-12, suggesting that ER
stress is involved in this disease.
Golgi stress response
The Golgi apparatus is another organelle responsible
for the production of secretory proteins [288]. Secre-
tory proteins synthesized in the ER are transported to
the Golgi apparatus and receive various modifications
by enzymes located there, such as the modification
of oligosaccharide chains and processing of peptide
chains. Proteins properly processed in the Golgi appar-
atus are transported to the plasma membrane or secre-
ted to the extracellular matrix. Thus, it is highly
possible that the Golgi apparatus sends an emergency
signal to the nucleus when the amount of client
exceeds the capacity of its processing machinery, indu-
cing the transcription of genes involved in Golgi
apparatus function [289]. Although the mechanism of
this response pathway (the Golgi apparatus stress
response pathway) has not yet been clarified, it is
worth investigating Golgi apparatus stress as it is
probably correlated with various diseases.
Conclusions and perspectives

Clarification of the basic mechanisms of ER stress
response has led to the discovery of a close relation-
ship between ER stress and various diseases. As
research on ER stress has expanded explosively, I hope
this small review will help to give researchers a com-
prehensive view and to further develop this field aca-
demically and clinically.
Acknowledgements
We thank Ms. Kaoru Miyagawa for secretarial assist-
ance. This work was supported by the PRESTO-
SORST program of the Japan Science and Technology
Agency, and grants from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan
(17026022, 17370061 and 18050013).
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